During retinal development, the decision to exit the cell cycle must be precisely coordinated with cell fate specification and differentiation to ensure that the correct proportion of each cell type is generated. The Rb family of proteins (Rb, p107 and p130) regulate cell cycle exit, cell fate specification, differentiation and survival during development. In the previous funding period, we focused on the unique and overlapping roles of the Rb family in regulating retinal progenitor cell proliferation during development in mice and humans. These studies moved the field forward and allowed us to develop some of the first knockout mouse models of retinoblastoma. We used these and other preclinical models of retinoblastoma to test new therapies and our research has directly impacted an ongoing clinical trial (RET-5) at St. Jude. In this grant proposal, we will extend our previous studies and focus on the role of the Rb family in neuronal cell fate specification and differentiation. Our preliminary data suggest that Rb regulates these two processes through distinct mechanisms. Specifically, we propose that Rb regulates rod photoreceptor cell fate specification by repressing aE2Fs on the Chx10 and Pax6 promoters and Rb regulates rod photoreceptor differentiation through rE2Fs and histone modification to activate the Nrl, Nr2e3 and Crx promoters. This is the first example of such complex regulation of neurogenesis by a tumor suppressor. In contrast to rod photoreceptors, horizontal neurons in the developing retina do not require the Rb family for cell fate specification, migration or differentiation. However, in the absence of the Rb family, mature horizontal cells re-enter the cell cycle and form metastatic retinoblastoma while maintaining their differentiated state. These data challenge the widely held belief in developmental biology that differentiation and proliferation are incompatible in neurons. More importantly they suggest that cells that rely upon Rb for their normal development such as rod photoreceptors are resistant to tumor formation following Rb family inactivation but cells that do not rely upon the Rb family for their normal development such as horizontal cells are more susceptible to tumorigenesis following Rb family gene inactivation. We will determine here if the role of the Rb family during normal development in different cell types directly influences their susceptibility to become tumorigenic.